Production of the Catechol Type Siderophore Bacillibactin by the Honey Bee Pathogen Paenibacillus larvae

The Gram-positive bacterium Paenibacillus larvae is the etiological agent of American Foulbrood. This bacterial infection of honey bee brood is a notifiable epizootic posing a serious threat to global honey bee health because not only individual larvae but also entire colonies succumb to the disease. In the recent past considerable progress has been made in elucidating molecular aspects of host pathogen interactions during pathogenesis of P. larvae infections. Especially the sequencing and annotation of the complete genome of P. larvae was a major step forward and revealed the existence of several giant gene clusters coding for non-ribosomal peptide synthetases which might act as putative virulence factors. We here present the detailed analysis of one of these clusters which we demonstrated to be responsible for the biosynthesis of bacillibactin, a P. larvae siderophore. We first established culture conditions allowing the growth of P. larvae under iron-limited conditions and triggering siderophore production by P. larvae. Using a gene disruption strategy we linked siderophore production to the expression of an uninterrupted bacillibactin gene cluster. In silico analysis predicted the structure of a trimeric trithreonyl lactone (DHB-Gly-Thr)3 similar to the structure of bacillibactin produced by several Bacillus species. Mass spectrometric analysis unambiguously confirmed that the siderophore produced by P. larvae is identical to bacillibactin. Exposure bioassays demonstrated that P. larvae bacillibactin is not required for full virulence of P. larvae in laboratory exposure bioassays. This observation is consistent with results obtained for bacillibactin in other pathogenic bacteria

Métodos para la investigación de la loque americana

American foulbrood is one of the most devastating diseases of the honey bee. It is caused by the spore-forming, Gram-positive rod-shaped bacterium Paenibacillus larvae. The recent updated genome assembly and annotation for this pathogen now permits in-depth molecular studies. In this paper, selected techniques and protocols for American foulbrood research are provided, mostly in a recipe-like format that permits easy implementation in the laboratory. Topics covered include: working with Paenibacillus larvae, basic microbiological techniques, experimental infection, and “’omics” and other sophisticated techniques. Further, this chapter covers other technical information including biosafety measures to guarantee the safe handling of this pathogen.La loque americana es una de las enfermedades más devastadoras de la abeja melífera, causada por el bacilo, formador de esporas Grampositivo Paenibacillus larvae. El reciente ensamblaje y anotación del genoma de este patógeno permite actualmente la realización de profundos estudios moleculares. En este trabajo, se proporcionan técnicas y protocolos seleccionados para la investigación de la loque americana, principalmente bajo la forma de protocolos de trabajo con una estructura similar al de las recetas, para facilitar su implementación en el laboratorio. Los temas desarrollados incluyen: el trabajo con Paenibacillus larvae, técnicas básicas microbiológicas, la infección experimental, y "'ómicas" y otras técnicas sofisticadas. Además, este capítulo abarca otro tipo de información técnica, incluyendo medidas de bioseguridad para garantizar la seguridad en el manejo de este patógeno.Trabajo publicado en Dietemann, V.; Ellis, J. D.; Neumann, P. (eds.) The Coloss Beebook, Volume II: standard methods for Apis mellifera pest and pathogen research. Journal of Apicultural Research, 52(1).Facultad de Ciencias Agrarias y Forestale

A taxonomic backbone for the global synthesis of species diversity in the angiosperm order Caryophyllales

The Caryophyllales constitute a major lineage of flowering plants with approximately 12500 species in 39 families. A taxonomic backbone at the genus level is provided that reflects the current state of knowledge and accepts 749 genera for the order. A detailed review of the literature of the past two decades shows that enormous progress has been made in understanding overall phylogenetic relationships in Caryophyllales. The process of re-circumscribing families in order to be monophyletic appears to be largely complete and has led to the recognition of eight new families (Anacampserotaceae, Kewaceae, Limeaceae, Lophiocarpaceae, Macarthuriaceae, Microteaceae, Montiaceae and Talinaceae), while the phylogenetic evaluation of generic concepts is still well underway. As a result of this, the number of genera has increased by more than ten percent in comparison to the last complete treatments in the Families and genera of vascular plants” series. A checklist with all currently accepted genus names in Caryophyllales, as well as nomenclatural references, type names and synonymy is presented. Notes indicate how extensively the respective genera have been studied in a phylogenetic context. The most diverse families at the generic level are Cactaceae and Aizoaceae, but 28 families comprise only one to six genera. This synopsis represents a first step towards the aim of creating a global synthesis of the species diversity in the angiosperm order Caryophyllales integrating the work of numerous specialists around the world

Neuromodulator release in neurons requires two functionally redundant calcium sensors

Neuropeptides and neurotrophic factors secreted from dense core vesicles (DCVs) control many brain functions, but the calcium sensors that trigger their secretion remain unknown. Here, we show that in mouse hippocampal neurons, DCV fusion is strongly and equally reduced in synaptotagmin-1 (Syt1)- or Syt7-deficient neurons, but combined Syt1/Syt7 deficiency did not reduce fusion further. Cross-rescue, expression of Syt1 in Syt7-deficient neurons, or vice versa, completely restored fusion. Hence, both sensors are rate limiting, operating in a single pathway. Overexpression of either sensor in wild-type neurons confirmed this and increased fusion. Syt1 traveled with DCVs and was present on fusing DCVs, but Syt7 supported fusion largely from other locations. Finally, the duration of single DCV fusion events was reduced in Syt1-deficient but not Syt7-deficient neurons. In conclusion, two functionally redundant calcium sensors drive neuromodulator secretion in an expression-dependent manner. In addition, Syt1 has a unique role in regulating fusion pore duration